Novel phylogenetic clade of avian Haemoproteus parasites (Haemosporida, Haemoproteidae) from Accipitridae raptors, with description of a new Haemoproteus species.

Avian haemosporidian parasites (order Haemosporida, phylum Apicomplexa) are blood and tissue parasites transmitted by blood-sucking dipteran insects. Three genera (Plasmodium, Haemoproteus and Leucocytozoon) have been most often found in birds, with over 270 species described and named in avian hosts based mainly on the morphological characters of blood stages. A broad diversity of Haemoproteus parasites remains to be identified and characterized morphologically and molecularly, especially those infecting birds of prey, an underrepresented bird group in haemosporidian parasite studies. The aim of this study was to investigate and identify Haemoproteus parasites from a large sample comprising accipitriform raptors of 16 species combining morphological and new molecular protocols targeting the cytb genes of this parasite group. This study provides morphological descriptions and molecular characterizations of two Haemoproteus species, H. multivacuolatus n. sp. and H. nisi Peirce and Marquiss, 1983. Haemoproteus parasites of this group were so far found in accipitriform raptors only and might be classified into a separate subgenus or even genus. Cytb sequences of these parasites diverge by more than 15% from those of all others known avian haemosporidian genera and form a unique phylogenetic clade. This study underlines the importance of developing new diagnostic tools to detect molecularly highly divergent parasites that might be undetectable by commonly used conventional tools.

Title: Nouveau clade phylogénétique de parasites de rapaces Accipitridae du genre Haemoproteus (Haemosporida, Haemoproteidae), avec description d'une nouvelle espèce d'Haemoproteus. Abstract: Les parasites hémosporidies aviaires (ordre Haemosporida, phylum Apicomplexa) sont des parasites sanguins et tissulaires transmis par des insectes diptères hématophages. Trois genres (Plasmodium, Haemoproteus et Leucocytozoon) ont été le plus souvent trouvés chez les oiseaux, avec plus de 270 espèces décrites et nommées chez les hôtes aviaires en fonction principalement des caractères morphologiques des stades sanguins. Une grande diversité des Haemoproteus reste à identifier et à caractériser morphologiquement et génétiquement, en particulier ceux qui infectent les oiseaux de proie, un groupe d'oiseaux sous-représenté dans les études sur les hémosporidies. Le but de cette étude était d'étudier et d'identifier les Haemoproteus à partir d'un large échantillon comprenant des rapaces accipitriformes de 16 espèces, en combinant des protocoles morphologiques et de nouveaux protocoles moléculaires ciblant les gènes cytb de ce groupe de parasites. Cette étude fournit des descriptions morphologiques et des caractérisations moléculaires de deux espèces d'Haemoproteus, H. multivacuolatus n. sp. et H. nisi Peirce and Marquiss, 1983. Les Haemoproteus de ce groupe n'ont jusqu'à présent été trouvés que chez les rapaces accipitriformes et pourraient être classés dans un sous-genre ou même un genre distinct. Les séquences cytb de ces parasites divergent de plus de 15 % de celles de tous les autres genres d'hémosporidies aviaires connus et forment un clade phylogénétique unique. Cette étude souligne l'importance de développer de nouveaux outils de diagnostic pour détecter des parasites moléculairement très divergents qui pourraient être indétectables par les outils conventionnels couramment utilisés.

Visceral Haemoproteus minutus Infection in a Major Mitchell's Cockatoo (Lophochroa leadbeateri).

A 1-year-old major Mitchell's cockatoo (Lophochroa leadbeateri) was presented for evaluation of weakness, diarrhea with undigested seeds in the droppings, and weight loss. Leukocytosis with severe heterophilia, monocytosis, and lymphocytosis was noted on the complete blood count. Altered plasma biochemical parameters included a slight increase in creatine kinase and mild hypoproteinemia. Two blood smears before and after 2 days of treatment revealed mild polychromasia and anisocytosis but no blood parasites. Radiographic and computed tomographic imaging of the cockatoo were helpful in identifying airsacculitis, pneumonia, and gastrointestinal motility disorders. The patient died 5 days after treatment for the presenting clinical problems. On the gross postmortem examination, dark red foci in the ventricular muscle layers and 1-3-mm white foci in the myocardium, opaque air sacs, and dark lungs were identified. Histopathologic examination of submitted tissue samples found severe granulomatous ventriculitis and myocarditis with intralesional Haemoproteus species megalomeronts. Qualitative polymerase chain reaction testing for the cytochrome b (cyt b) gene performed on pooled heart, liver, kidney, and intestinal tissues identified 99.5% homology to Haemoproteus minutus. This case report demonstrates the expansion of the geographic range of H minutus to France and potentially to Belgium, which may compromise breeding and conservation of Australian parrots living outdoors. Challenging diagnosis, rapid disease progression, and the absence of validated treatment protocols for psittacine patients suggest that the use of preventive measures to reduce the presence of insect vectors such as hippoboscid flies and biting midges (Culicoides) should be considered. Haemoproteus minutus should be considered and potentially screened by polymerase chain reaction testing on blood samples, especially in the case of highly susceptible avian species (eg, Australian parrots in Europe) that present with sudden weakness, heterophilic leukocytosis, and monocytosis associated with mild anemia.

Marine gregarine genomes reveal the breadth of apicomplexan diversity with a partially conserved glideosome machinery.

Our current view of the evolutionary history, coding and adaptive capacities of Apicomplexa, protozoan parasites of a wide range of metazoan, is currently strongly biased toward species infecting humans, as data on early diverging apicomplexan lineages infecting invertebrates is extremely limited. Here, we characterized the genome of the marine eugregarine Porospora gigantea, intestinal parasite of Lobsters, remarkable for the macroscopic size of its vegetative feeding forms (trophozoites) and its gliding speed, the fastest so far recorded for Apicomplexa. Two highly syntenic genomes named A and B were assembled. Similar in size (~ 9 Mb) and coding capacity (~ 5300 genes), A and B genomes are 10.8% divergent at the nucleotide level, corresponding to 16-38 My in divergent time. Orthogroup analysis across 25 (proto)Apicomplexa species, including Gregarina niphandrodes, showed that A and B are highly divergent from all other known apicomplexan species, revealing an unexpected breadth of diversity. Phylogenetically these two species branch sisters to Cephaloidophoroidea, and thus expand the known crustacean gregarine superfamily. The genomes were mined for genes encoding proteins necessary for gliding, a key feature of apicomplexans parasites, currently studied through the molecular model called glideosome. Sequence analysis shows that actin-related proteins and regulatory factors are strongly conserved within apicomplexans. In contrast, the predicted protein sequences of core glideosome proteins and adhesion proteins are highly variable among apicomplexan lineages, especially in gregarines. These results confirm the importance of studying gregarines to widen our biological and evolutionary view of apicomplexan species diversity, and to deepen our understanding of the molecular bases of key functions such as gliding, well known to allow access to the intracellular parasitic lifestyle in Apicomplexa.

Haemosporidian parasites from captive Strigiformes in France.

Haemosporidia infections may cause major damage to avian populations and represent a concern for veterinarians working in zoological parks or wildlife rescue centres. Following the fatal infection of 9 Great grey owls (Strix nebulosa) at Mulhouse zoological park, between summer 2013 and 2015, a prospective epidemiological investigation was performed in captive strigiform birds in France in 2016. The purpose was to evaluate the prevalence of haemosporidian parasites in captive Strigiformes and to estimate the infection dynamics around the nesting period. Blood samples were taken from 122 strigiform birds representing 14 species from 15 French zoological parks. Parasites were detected by direct examination of blood smears and by PCR targeting the mitochondrial cytochrome b gene. Haemosporidian parasites were detected in 59 birds from 11 zoos. Three distinct Haemoproteus mitochondrial cytochrome b sequences (haplotypes A and C for H. syrnii and haplotype B for Haemoproteus sp.) as well as two species of Plasmodium were detected. The overall prevalence of Haemoproteus infection was 12.8%. The percentage of birds infected by Haemoproteus varied according to the period of sampling. Nesting season seemed to be at greater risk with an average prevalence of 53.9% compared with winter season with an average prevalence of 14.8%, related to the abundance of the vectors. The prevalence of Plasmodium infection in Strigiformes did not exceed 8% throughout the year. This study confirmed how significant Haemosporidia infection could be in Strigiformes from zoological parks in France. The nesting season was identified as a period of higher risk of infection and consequently the appropriate period to apply prophylactic measures.

Reducing blood stream infection in patients on hemodialysis: Incorporating patient engagement into a quality improvement activity.

Introduction Infection in our immunocompromised patients is the second leading cause of death, according to the Centers for Disease Control and Prevention (CDC). In an effort to improve quality of care, engage patients in their own care, and reduce morbidity and mortality secondary to infection, the Network designed a joint quality improvement/patient engagement activity to decrease bloodstream infection (BSI) rates. Methods Dialysis facilities were ranked utilizing 2014 National Healthcare Safety Network (NHSN) data. Selection included 20% of Network 13 facilities (n = 58) with the highest BSI rates, which captured 31% of the patient population. Findings Statistically significant (P < 0.001) improvement was reached in the reduction of BSIs; increasing patient engagement in the infection control process; and, correct completion of hand hygiene audits. Significant (P < 0.01) improvement was reached in correct completion of cannulation audits. There was also improvement in the catheter audits, but results were not significant. Discussion Involving patients in the infection control process contributed to our successful outcomes and could be replicated to meet the needs of the end stage renal disease community as a whole.

Highly rearranged mitochondrial genome in Nycteria parasites (Haemosporidia) from bats.

Haemosporidia parasites have mostly and abundantly been described using mitochondrial genes, and in particular cytochrome b (cytb). Failure to amplify the mitochondrial cytb gene of Nycteria parasites isolated from Nycteridae bats has been recently reported. Bats are hosts to a diverse and profuse array of Haemosporidia parasites that remain largely unstudied. There is a need to obtain more molecular data from chiropteran parasites. Such data would help to better understand the evolutionary history of Haemosporidia, which notably include the Plasmodium parasites, malaria's agents. We use next-generation sequencing to obtain the complete mitochondrial genome of Nycteria parasites from African Nycteris grandis (Nycteridae) and Rhinolophus alcyone (Rhinolophidae) and Asian Megaderma spasma (Megadermatidae). We report four complete mitochondrial genomes, including two rearranged mitochondrial genomes within Haemosporidia. Our results open outlooks into potentially undiscovered Haemosporidian diversity.

Exploring the environmental diversity of kinetoplastid flagellates in the high-throughput DNA sequencing era

The class Kinetoplastea encompasses both free-living and parasitic species from a wide range of hosts. Several representatives of this group are responsible for severe human diseases and for economic losses in agriculture and livestock. While this group encompasses over 30 genera, most of the available information has been derived from the vertebrate pathogenic genera Leishmaniaand Trypanosoma. Recent studies of the previously neglected groups of Kinetoplastea indicated that the actual diversity is much higher than previously thought. This article discusses the known segment of kinetoplastid diversity and how gene-directed Sanger sequencing and next-generation sequencing methods can help to deepen our knowledge of these interesting protists.

Exploring the environmental diversity of kinetoplastid flagellates in the high-throughput DNA sequencing era.

The class Kinetoplastea encompasses both free-living and parasitic species from a wide range of hosts. Several representatives of this group are responsible for severe human diseases and for economic losses in agriculture and livestock. While this group encompasses over 30 genera, most of the available information has been derived from the vertebrate pathogenic genera Leishmaniaand Trypanosoma. Recent studies of the previously neglected groups of Kinetoplastea indicated that the actual diversity is much higher than previously thought. This article discusses the known segment of kinetoplastid diversity and how gene-directed Sanger sequencing and next-generation sequencing methods can help to deepen our knowledge of these interesting protists.

Haemoproteus ilanpapernai n. sp. (Apicomplexa, Haemoproteidae) in Strix seloputo from Singapore: morphological description and reassignment of molecular data.

Haemoproteus ilanpapernai Karadjian and Landau n. sp. from the Spotted Wood Owl, Strix seloputo, in Singapore is described from material from Ilan Paperna's collection of slides. The species was previously identified as Haemoproteus syrnii (Mayer, 1910). However, comparisons between the material from Strix seloputo and our own material from Strix aluco, the type host of H. syrnii, revealed morphological and molecular differences. H. ilanpapernai n. sp. differs morphologically from H. syrnii by the much smaller size of the gametocytes, the different position of the mature gametocytes in the erythrocyte (apical, subapical, or lateral in H. ilanpapernai vs. always lateral in H. syrnii), the effect on the erythrocyte nucleus (frequently tilted in H. ilanpapernai but not displaced laterally vs. straight and displaced laterally in H. syrnii) and characters of the pigment (aggregated in the gametocytes of H. ilanpapernai vs. dispersed in H. syrnii). A molecular analysis showed that the two species differ by 2.9% at the cyt b and 3.1% at the COI genes.

Haemoproteus syrnii in Strix aluco from France: morphology, stages of sporogony in a hippoboscid fly, molecular characterization and discussion on the identification of Haemoproteus species.

In France, Haemoproteus syrnii is frequently found in the Tawny Owl, Strix aluco. Additional and complementary features of this species, and in particular the characteristics of volutin, are presented. The authors consider the volutin granules as constant in a given species, and discuss their taxonomic value. These cytoplasmic inclusions appear early during the first stages of development of the gametocytes as an initial granule which multiplies as the parasite develops. They were reported in some species of Haemoproteus but are seldom considered as a specific character and described with precision. Sporogony from ookinete to apparently mature sporozoites appears to take place in a pupiparous hippoboscid (Ornithomyia sp.). One specimen was crushed between two slides and stained with Giemsa. Gametocytes of H. syrnii, many ookinetes, an immature oocyst and mature sporozoites were observed spread all over the smear. This would allow classifying this species in the Haemoproteus subgenus. We provide associated molecular data derived from the cyt b and cox 1 gene from this parasite. We discuss the problems of multiple infections and the difficulties in identifying Haemoproteus species and in deriving conclusions from sequences deposited in databases.

Nonhuman primate retroviruses from Cambodia: high simian foamy virus prevalence, identification of divergent STLV-1 strains and no evidence of SIV infection.

Nonhuman primates (NHPs) carry retroviruses such as simian immunodeficiency viruses (SIV), simian T-cell lymphotropic viruses (STLV) and simian foamy viruses (SFV). Here, we revisited NHPs from Cambodia to assess the prevalence and diversity of these retroviruses using updated viral detection tools. We screened blood from 118 NHPs consisting of six species (Macaca fascicularis (n=91), Macaca leonine (n=8), Presbytis cristata (n=3), Nycticebus coucang (n=1), Hylobates pileatus (n=14), and Pongo pygmaeus) (n=1) by using a Luminex-based multiplex serology assay that allows the detection of all known SIV/HIV and SFV lineages. We also used highly sensitive PCR assays to detect each simian retrovirus group. Positive PCR products were sequenced and phylogenetically analyzed to infer evolutionary histories. Fifty-three of 118 (44.9%) NHPs tested positive for SFV by serology and 8/52 (15.4%), all from M. fascicularis, were PCR-confirmed. The 8 novel SFV sequences formed a highly supported distinct lineage within a clade composed of other macaque SFV. We observed no serological or molecular evidence of SIV infection among the 118 NHP samples tested. Four of 118 (3.3%) NHPs were PCR-positive for STLV, including one M. fascicularis, one P. cristata, and two H. pileatus. Phylogenetic analyses revealed that the four novel STLV belonged to the PTLV-1 lineage, outside the African radiation of PTLV-1, like all Asian PTLV identified so far. Sequence analysis of the whole STLV-1 genome from a H. pileatus (C578_Hp) revealed a genetic structure characteristic of PTLV. Similarity analysis comparing the STLV-1 (C578_Hp) sequence with prototype PTLVs showed that C578_Hp is closer to PTLV-1s than to all other types across the entire genome. In conclusion, we showed a high frequency of SFV infection but found no evidence of SIV infection in NHPs from Cambodia. We identified for the first time STLV-1 in a P. cristata and in two H. pileatus.

Quality improvement project: hemodialysis vascular access management trends report.

Vascular access for the delivery of hemodialysis is the initial clinical procedure for any applicable HD patient. It behooves clinicians to ensure that these patients have a vascular access management plan assuring the best permanent vascular access possible for optimal patient care outcomes. AVFs are considered the preferred access based on their superior patency, minimal complication rates, and decreased risk of patient mortality. This project's activities continue to address practice barriers and motivate changes where necessary to improve patient care.

The chiropteran haemosporidian Polychromophilus melanipherus: a worldwide species complex restricted to the family Miniopteridae.

This paper attempts to expand on the current knowledge regarding the evolutionary history of bat haemosporidian parasites. Using modern molecular tools as adjuncts to existing morphological descriptions, our understanding of the diversity of these parasites is discussed. The biogeography and host range distribution together with possible host-parasite interactions remain to be evaluated in more detail. Using a nested-PCR cytochrome b mitochondrial gene approach, we established a screening programme and survey of several months duration for haemosporidian parasites in four central African bat species living in an ecological community. The aim of the study was to describe parasites morphologically and molecularly, together with parasite prevalence variations over time, and evaluate parasite host-specificity in these sympatric cave bats. Over the survey period, Polychromophilus melanipherus was the only haemosporidian parasite identified in Miniopterus inflatus, with a continuous molecular prevalence of at least 60%. Molecular phylogenetic analyses show that P. melanipherus is a monophyletic group infecting Miniopterus bats which is, a sister group to P. murinus and Polychromophilus spp. This monophyletic group is composed of different cyt b haplotypes molecularly distantly related (but morphologically similar), circulating without geographic or host species distinction. This suggests that P. melanipherus is a species complex restricted to the family Miniopteridae. The phylogenetic analysis confirms that Polychromophilus parasites are distributed worldwide and supports the view that they are more closely related to avian haemosporidian parasites.

Network 13 partnership to improve the influenza, pneumococcal pneumonia, and hepatitis B vaccination rates among dialysis patients.

Vaccinations are available for primary prevention of many infections in adults. Morbidity and mortality from invasive diseases such as influenza and Streptococcus pneumoniae (pneumococcus) remain high and may be largely preventable by vaccination of high-risk adults, including dialysis patients. The current 23-valent vaccine-efficacious, with a low adverse event profile-is widely available. Revaccination is also recommended in patients with immunocompromising conditions, including chronic kidney disease. Despite having many opportunities to be vaccinated, adult hemodialysis and peritoneal dialysis patients are often missed During the recent H1N1 influenza outbreak, we conducted a performance improvement project to increase the vaccination rates for pneumococcal pneumonia, hepatitis B, and influenza, with a special focus on prevention. The project included an education phase, baseline assessment of vaccination rates, intervention, and a follow-up assessment of vaccination rates. The geographic jurisdiction of ESRD Network 13 encompasses the states of Arkansas, Louisiana, and Oklahoma. At the beginning of the network-wide project, the documented state-specific rates for influenza immunization were below the average influenza immunization rates for adults reported by Centers for Disease Control and Prevention and far below its target for adults. Our improvement project incorporated educational interventions to improve patient acceptance of vaccinations, educational interventions to improve staff participation in quality improvement activities, and improved techniques of quality improvement data collection and analysis by participants. During this project, the immunization rates for hepatitis B and pneumococcal pneumonia were also reviewed. At project's conclusion, improvement was demonstrated in all three focus areas, with statistically significant improvements noted in both influenza and pneumococcus vaccination rates. The use of educational interventions to improve staff participation in quality improvement, and the collection and analysis of quality improvement data can be replicated in many practice settings to improve immunization rates for dialysis patients and other patients with chronic illnesses.

Plasmodium knowlesi infection in humans, Cambodia, 2007-2010.

Two cases of Plasmodium knowlesi infection in humans were identified in Cambodia by 3 molecular detection assays and sequencing. This finding confirms the widespread distribution of P. knowlesi malaria in humans in Southeast Asia. Further wide-scale studies are required to assess the public health relevance of this zoonotic malaria parasite.

A fresh look at the origin of Plasmodium falciparum, the most malignant malaria agent.

From which host did the most malignant human malaria come: birds, primates, or rodents? When did the transfer occur? Over the last half century, these have been some of the questions up for debate about the origin of Plasmodium falciparum, the most common and deadliest human malaria parasite, which is responsible for at least one million deaths every year. Recent findings bring elements in favor of a transfer from great apes, but are these evidences really solid? What are the grey areas that remain to be clarified? Here, we examine in depth these new elements and discuss how they modify our perception of the origin and evolution of P. falciparum. We also discuss the perspectives these new discoveries open.

Infection control 101.

Healthcare professionals in dialysis must provide safe, quality care, including the use of basic infection control practices when caring for immunocompromised patients on dialysis. These practices are not new, and for most, are integrated into the routine provision of dialysis care. The challenge within the existing time constraints is to make sure routine practices do not lapse into shortcuts around infection control practices. This article reviews basic infection control as a reminder of its importance in the daily provision of health care.

African apes as reservoirs of Plasmodium falciparum and the origin and diversification of the Laverania subgenus.

We investigated two mitochondrial genes (cytb and cox1), one plastid gene (tufA), and one nuclear gene (ldh) in blood samples from 12 chimpanzees and two gorillas from Cameroon and one lemur from Madagascar. One gorilla sample is related to Plasmodium falciparum, thus confirming the recently reported presence in gorillas of this parasite. The second gorilla sample is more similar to the recently defined Plasmodium gaboni than to the P. falciparum-Plasmodium reichenowi clade, but distinct from both. Two chimpanzee samples are P. falciparum. A third sample is P. reichenowi and two others are P. gaboni. The other chimpanzee samples are different from those in the ape clade: two are Plasmodium ovale, and one is Plasmodium malariae. That is, we have found three human Plasmodium parasites in chimpanzees. Four chimpanzee samples were mixed: one species was P. reichenowi; the other species was P. gaboni in three samples and P. ovale in the fourth sample. The lemur sample, provisionally named Plasmodium malagasi, is a sister lineage to the large cluster of primate parasites that does not include P. falciparum or ape parasites, suggesting that the falciparum + ape parasite cluster (Laverania clade) may have evolved from a parasite present in hosts not ancestral to the primates. If malignant malaria were eradicated from human populations, chimpanzees, in addition to gorillas, might serve as a reservoir for P. falciparum.

Sub-microscopic malaria cases and mixed malaria infection in a remote area of high malaria endemicity in Rattanakiri province, Cambodia: implication for malaria elimination.

BACKGROUND: Malaria microscopy and rapid diagnostic tests are insensitive for very low-density parasitaemia. This insensitivity may lead to missed asymptomatic sub-microscopic parasitaemia, a potential reservoir for infection. Similarly, mixed infections and interactions between Plasmodium species may be missed. The objectives were first to develop a rapid and sensitive PCR-based diagnostic method to detect low parasitaemia and mixed infections, and then to investigate the epidemiological importance of sub-microscopic and mixed infections in Rattanakiri Province, Cambodia. METHODS: A new malaria diagnostic method, using restriction fragment length polymorphism analysis of the cytochrome b genes of the four human Plasmodium species and denaturing high performance liquid chromatography, has been developed. The results of this RFLP-dHPLC method have been compared to 1) traditional nested PCR amplification of the 18S rRNA gene, 2) sequencing of the amplified fragments of the cytochrome b gene and 3) microscopy. Blood spots on filter paper and Giemsa-stained blood thick smears collected in 2001 from 1,356 inhabitants of eight villages of Rattanakiri Province have been analysed by the RFLP-dHPLC method and microscopy to assess the prevalence of sub-microscopic and mixed infections. RESULTS: The sensitivity and specificity of the new RFLP-dHPLC was similar to that of the other molecular methods. The RFLP-dHPLC method was more sensitive and specific than microscopy, particularly for detecting low-level parasitaemia and mixed infections. In Rattanakiri Province, the prevalences of Plasmodium falciparum and Plasmodium vivax were approximately two-fold and three-fold higher, respectively, by RFLP-dHPLC (59% and 15%, respectively) than by microscopy (28% and 5%, respectively). In addition, Plasmodium ovale and Plasmodium malariae were never detected by microscopy, while they were detected by RFLP-dHPLC, in 11.2% and 1.3% of the blood samples, respectively. Moreover, the proportion of mixed infections detected by RFLP-dHPLC was higher (23%) than with microscopy (8%). CONCLUSIONS: The rapid and sensitive molecular diagnosis method developed here could be considered for mass screening and ACT treatment of inhabitants of low-endemicity areas of Southeast Asia.

An arteriovenous fistula (AVF) functionality quality improvement tracking tool--developed and implemented to improve newly placed AVF utilization rates and patient outcomes.

The authors developed a reporting tool to assist hemodialysis clinicians to track new arteriovenous fistulas (AVFs), their maturation, and use. The tool identifies impediments to timely use (6 weeks/42 days) of AVFs. The use of this tool in nine dialysis units with high gaps between AVF placement and usage reduced the gap from 19.5% to 13.5% and reflected a reduction in the percentage of AVFs in place but not in use from 31.4% to 23.2%.